TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model. Drug discovery has traditionally involved a search for inhibitors of protein complexation to small molecules (e.g., substrates or ligands) or macromolecules (e.g., other proteins, nucleic acids, or polysaccharides). While agonists or antagonists of macromolecular receptors that become useful drugs must display a wide range of other attributes, including the appropriate physicochemical properties, pharmacokinetic and pharmacodynamic properties, stability, etc., they must first be protein ligands. The vast majority of available drugs act by binding noncovalently to a protein, preventing or (less often) stimulating that protein's complexation to a complement (1).Complexes of nucleic acid and proteins are key intermediates in all transcriptional and translational processes. Some nucleic acids (e.g., ribozymes) even form functional complexes with small molecules (2). Nonetheless, nucleic acids are widely viewed as ineffective targets for the discovery of low-molecular weight inhibitors. One reason is that the linear motif in single-stranded DNA and the repetitive motif in double-stranded DNA provide attractive targets for large, linear binding molecules (3), but unattractive targets for the small molecules that lead to orally available medications. Another reason is that the lack of tertiary structure in DNA does not afford the diverse topology associated with folded proteins.However, single-stranded RNA often folds into welldefined tertiary structures (4). Furthermore, such structures serve as docking sites for transcriptional activators (5) and substrates for self-splicing reactions (6, 7). Can such structured nucleic acids display sufficient shape diversity to permit the complexation of a small molecule at one site in a virtual sea of nucleic acid material? This is the essential question whose presumed negative answer hinders drug discovery at the nucleic acid level.Certain cis-acting RNA elements are essential for the gene expression of human immunodeficiency virus type 1 (HIV-1) (8). The functions and sequences of these RNA molecules have been well characterized (9-11). A segment of HIV-1 mRNA (residues 1-59), identified as the transactivation responsive element (TAR), adopts a stem-loop secondary structure consisting of a highly cons...
The paradigm for the control of feeding behavior has changed significantly. In this review, we present evidence that the separation of function in which cholecystokinin (CCK) controls short-term food intake and leptin regulate long-term eating behavior and body weight become less clear. In addition to the hypothalamus, the vagus nerve is critically involved in the control of feeding by transmitting signals arising from the upper gut to the nucleus of the solitary tract. Among the peripheral mediators, CCK is the key peptide involved in generating the satiety signal via the vagus. Leptin receptors have also been identified in the vagus nerve. Studies in the rodents clearly indicate that leptin and CCK interact synergistically to induce short-term inhibition of food intake and long-term reduction of body weight. The synergistic interaction between vagal CCK-A receptor and leptin is mediated by the phosphorylation of signal transducer and activator of transcription3 (STAT3), which in turn, activates closure of K+ channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/SRC/phosphoinositide 3-kinase cascades and leptin/Janus kinase-2/phosphoinositide 3-kinase/STAT3 signaling pathways. It is conceivable that malfunctioning of these signaling molecules may result in eating disorders.
Research has shown that the synergistic interaction between vagal cholecystokinin-A receptors (CCKARs) and leptin receptors (LRbs) mediates short term satiety. We hypothesize that this synergistic interaction is mediated by cross-talk between signaling cascades used by CCKARs and LRbs, which, in turn, activates closure of K ؉ channels, leading to membrane depolar- Leptin, the product of the ob gene, is secreted primarily from white adipocyte tissue; its level in the circulation correlates with the degree of adiposity (1, 2). Circulating leptin crosses the blood-brain barrier via a receptor-mediated transport system (3, 4) and acts on the long form of the leptin receptor (LRb) 2 in the medial hypothalamus to regulate feeding behavior and energy balance (5). Leptin is secreted from several other sites, including the gastric mucosa, brown adipocyte tissue, placenta, mammary gland, ovarian follicles, and brain (5, 6). Leptin mRNA and leptin protein have also been detected in human stomach mucosa (7) and rat gastric fundus (8). Leptin levels in the stomach are altered by nutritional state and by cholecystokinin (CCK) administration. CCK is not, however, a stimulus for leptin release from isolated adipocytes (8). Leptin is the key signaling molecule responsible for long term satiety and energy balance; mutations that cause defective leptin secretion or abnormal leptin receptor signaling result in obesity in ob/ob mice (9, 10) and in humans (11). The leptin receptor belongs to the IL-6 receptor family of class 1 cytokine receptors and mediates the biological effects of leptin via the Janus kinase 2-signal transducer and activator of transcription 3 (JAK2/STAT3) pathway (12-14). Several splice variants of the leptin receptor exist; however, the LRb isoform mediates the leptin effect on satiety (4). CCK is an endogenous peptide found in the gastrointestinal tract and the brain. It is released into the circulation after a meal and acts on neurons both centrally and peripherally (15). The satiety action of CCK appears to be mediated by low affinity CCK-A receptors (CCKARs) on vagal afferent neurons (16). Systemic administration of CCK inhibits food intake in several species, including rats and humans (17), giving credence to the hypothesis that peripheral CCK acts as a satiety signal. CCK cannot penetrate the blood-brain barrier; therefore, systemically administered CCK likely acts at a peripheral site to inhibit feeding (18). In contrast to leptin, the effect of CCK on food intake occurs within 15 min after intraperitoneal administration of CCK-8, suggesting that CCK may act as a meal-related short term satiety signal (19,20).Both CCKARs and LRbs are widely distributed in nodose ganglia (NG) and the vagus nerve (21,22). There is evidence that a synergistic interaction between leptin and CCK leads to the reduction of short term food intake (23)(24)(25). In fact, the satiety action of CCK appears to depend on leptin signaling (26). Currently, the intracellular signaling mechanisms responsible for the synergistic interacti...
OBJECTIVEDiabetic patients often experience visceral hypersensitivity and anorectal dysfunction. We hypothesize that the enhanced excitability of colon projecting dorsal root ganglia (DRG) neurons observed in diabetes is caused by a decrease in the amplitude of the transient A-type K+ (IA) currents resulting from increased phosphorylation of mitogen-activated protein kinases (MAPK) and reduced opening of Kv4.2 channels.RESEARCH DESIGN AND METHODSWe performed patch-clamp recordings of colon projecting DRG neurons from control and streptozotocin-induced diabetic (STZ-D) rats. Western blot analyses and immunocytochemistry studies were used to elucidate the intracellular signaling pathways that modulate the IA current. In vivo studies were performed to demonstrate that abnormal MAPK signaling is responsible for the enhanced visceromotor response to colorectal distention in STZ-D rats.RESULTSPatch-clamp studies demonstrated that IA current was diminished in the colon projecting DRG neurons of STZ-D rats. Western blot analysis of STZ-D DRG neurons revealed increases in phosphorylated MAPK and KV4.2. In diabetic DRG neurons, increased intracellular Ca2+ ([Ca2+]i), protein kinase C (PKC), and MAPK were involved in the regulation of IA current through modulation of Kv4.2. Hypersensitive visceromotor responses to colorectal distention in STZ-D rats were normalized by administration of MAPK inhibitor U0126.CONCLUSIONSWe demonstrated that reduction of the IA current in STZ-D DRG neurons is triggered by impaired [Ca2+]i ion homeostasis, and this in turn activates the PKC-MAPK pathways, resulting in decreased opening of the Kv4.2 channels. Hence, the PKC-MAPK–Kv4.2 pathways represent a potential therapeutic target for treating visceral hypersensitivity in diabetes.
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